The simplest way to separate these peptides is by ion-exchange. With such
vastly differing pI values you can virtually use any ion exchanger and use
conditions where one peptide will stcik and the other won't. Simple.
Just a general comment (don't take it personally).......many younger
researchers have only ever association HPLC with reversed phase
separations. The selectivity, and indeed the huge loading capacity, of ion
exchange chromatography is often overlooked for peptides, but I've found it
to be an extremely useful, complementary technique, to be followed perhaps
by RP-HPLC to buffer exchange into a voltile solvent system.
Maybe I'm showing my age, but I would recommend to all young researchers to
look at ion exchange very closely....
Cheers,
Roger
At 04:59 PM 16-09-99 +0100, you wrote:
>
>
>Hello all,
>
>I'm trying to use reversed-phase HPLC to seperate and quantify an acidic
>(A) peptide and a basic (B) peptide. The A peptide is only soluble > pH
>6ish, but at this pH the B peptide sticks irretrievably to the column
>(Vydac TP218, C18).
>
>I presume it's getting stuck to silanol groups, as it is very basic,
>definitely soluble, and elutes fine at pH2. I also suspect that although
>changing my buffer system (Phosphate/MeCN) may help, it won't completely
>negate the problem, meaning that I'm still not getting quantitative
>results.
>
>>From reading the archives, I suspect a base-deactivated column is what I
>need. I can't find anything specifically labelled such in our catalogues -
>are there other terms for the same thing, or is it fairly specialised?
>Does it cost loads more?
>
>Alternatively, is there a way to solve my silanol problem without buying a
>new column? Or is there a completely different way to seperate these very
>differently charged peptides (PI's about 3 & 10) quantitively?
>
>Thanks!
>
>Jenny Shipway
>Centre for Biomolecular Drug Design and Drug Development
>University of Sussex
------------------------------------------------------------
Roger Murphy, Ph.D.
Biological Production Facility
Ludwig Institute for Cancer Research
Austin & Repatriation Medical Centre
Studley Road,
Heidelberg, Vic. 3084
Australia.
Tel 61-3-94965463
Fax 61-3-94965436
Email Roger.Murphy@Ludwig.edu.au